PROJECT SUMMARY PD1 and LAG3 both play critical roles in controlling T cell tolerance, preventing autoimmunity, and limiting immune-mediated tissue damage. Combined genetic deficiency of PD1 and LAG3 in mice results in lethal systemic autoimmunity, demonstrating critical PD1/LAG3 synergies in tolerance. PD1 and LAG3 also are mediators of T cell exhaustion, and synergize to limit viral or tumor control. PD1 and LAG3 are promising therapeutic targets, but little is know about their mechanisms of action or how synergy is mediated. Project 1 will investigate mechanisms by which PD1 and LAG3 interactions regulate the balance between pathogenic and protective immune responses in autoimmunity, using the experimental autoimmune encephalomyelitis (EAE) and type 1 diabetes models. Our overall hypothesis is that PD1 and LAG3 have synergistic effects in tolerance and autoimmunity by controlling functions of self-reactive CD4+ FoxP3- T cells and protective FoxP3+ Treg cells. We will use novel conditional PD1 and LAG3 knockout mice crossed to cell type-specific Cre lines to test this hypothesis: Aim 1: What are the relative roles of PD1 and LAG3 in controlling the generation and function of Treg cells? We will test the hypothesis that interactions between PD1 and LAG3 control Treg function and stability, and thereby impact self tolerance. Surprisingly, our preliminary data show that mice lacking LAG3 and/or PD1 in Treg develop ameliorated autoimmune disease. We will analyze the relative roles of PD1 and LAG3 in Treg, and determine how they contribute to Treg function in EAE and NOD diabetes. We will examine if there are interactions between LAG3/PD1 in the same or distinct Treg or between PD1/LAG3 on Treg and other cell types, and determine molecular pathways associated with LAG3/PD1 interactions in Treg during autoimmunity. Aim 2: What are the relative and synergistic roles of PD1 and LAG3 in regulating self-reactive CD4+ T cells? In contrast to ameliorated autoimmunity in mice lacking PD1 and/or LAG3 on Treg, loss of LAG3 and/or PD1 on all T cells accelerates autoimmunity. These disparate phenotypes lead us to hypothesize that PD1 and LAG3 synergize to have a dominant effect on CD4+ T cells in mediating tolerance to self antigens. To understand these differences, separate analyses of Treg and FoxP3- T cells are needed. In this aim we will determine the relative contributions of PD1 and LAG3 in regulating self-reactive CD4+ T cells. We will determine if there are synergies between LAG3/PD1 in CD4+ cells, if LAG3 and PD1 operate in the same or distinct CD4+ T cells, and molecular pathways associated with interactions between LAG3/PD1 in CD4+ T cells in autoimmune disease. Project 1 will collaborate with Project 2 to compare the impact of disruption of PD1/LAG3 signals in Treg in autoimmunity and cancer, and Project 3 to compare PD1/LAG3 interactions in autoreactive and exhausted CD4+ T cells. Project 1 will use Core B for mice, Core C for transcriptional analyses, Core D for immunopathology studies, and Core A for statistical support.